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Abstract

Background

Pulmonary adenocarcinomas with a micropapillary component having small papillary tufts
and lacking a central fibrovascular core are thought to result in poor prognosis.
However, the component consists of tumor cells often floating within alveolar spaces
(aerogenous micropapillary component [AMPC]) rather than invading fibrotic stroma
observed in other organs like breast (stromal invasive micropapillary component [SMPC]).
We previously observed cases of lung adenocarcinoma with predominant SMPC that was
associated with micropapillary growth of tumors in fibrotic stroma observed in other
organs. We evaluated the incidence and clinicopathological characteristics of SMPC
in lung adenocarcinoma cases.

Patients and Methods

We investigated the clinicopathological characteristics and prognostic significance
of SMPC in lung adenocarcinoma cases by reviewing 559 patients who had undergone surgical
resection. We examined the SMPC by performing immunohistochemical analysis with 17
antibodies and by genetic analysis with epidermal growth factor receptor (EGFR) and KRAS mutations.

Keywords:

Background

A new lung adenocarcinoma classification system has been proposed by the International
Association for the Study of Lung Cancer, American Thoracic Society, and European
Respiratory Society (IASLC/ATS/ERS) [1]. In this classification, the micropapillary component (MPC) was recommended as a
new subtype of lung adenocarcinoma in addition to the lepidic, acinar, papillary,
and solid subtypes defined in the 2004 World Health Organization (WHO) classification
[2]. MPC was defined as tumor cells growing in papillary tufts lacking fibrovascular
cores and may float within alveolar spaces. MPC-predominant lung adenocarcinoma shows
a high incidence of nodal metastasis and a poor prognosis [3-8]. MPC-predominant carcinomas developing in various other organs, such as the breast
and urinary bladder, known as invasive micropapillary carcinoma, also have a poor
prognosis. However, localization of MPC in the lungs is significantly different from
that in the other organs; MPC in lung adenocarcinoma is distinguished by floating
tumor cells within alveolar spaces (aerogenous micropapillary component, AMPC), while
MPC in other organs has been observed primarily in the stroma as invasive components
(stromal invasive micropapillary component, SMPC) [3,4].

Few studies have examined lung adenocarcinoma with SMPC [9,10]. Recently, we reported 2 cases of SMPC-predominant lung adenocarcinoma [9]. The proportion of SMPC in both tumors was greater than 50% in area. We observed
that SMPC had a strong association with vascular invasion, similar to the cases of
SMPC-predominant carcinoma in other organs. However, a large-scale investigation on
pulmonary SMPC has not been conducted.

The aims of this study included: (1) clarifying the incidence of SMPC in lung adenocarcinoma;
(2) elucidating the clinicopathological characteristics of the tumor; and (3) determining
the prognoses of the SMPC-positive (SMPC(+)) tumors and comparing them with those
of SMPC-negative (SMPC(-)) tumors. We reviewed 559 resected lung adenocarcinomas for
this study with performing immunohistochemical and genetic analysis.

Methods

Patients

We analyzed 565 consecutive cases of primary lung adenocarcinoma treated by surgical
resection at the Kanagawa Cancer Center between February 2007 and December 2010. Formalin
fixation of the resected lung tissue was performed within 48 hours to reduce the loss
of immunohistochemical antigen expression and degeneration of DNA. Six patients who
had received preoperative chemotherapy were excluded. A total of 559 cases were enrolled
in the study. The median follow-up time was 634.5 days (range, 28-1512 days). All
patients provided informed consent, and the studies were performed according to the
requirements of the institutional review board of Kanagawa Cancer Center.

Pathological review

Excised specimens were fixed in a solution of 10% buffered formaldehyde, and the sections
were embedded in paraffin. Next, 4-μm-thick sections, including the largest cut surface
of the tumor, were prepared and stained using hematoxylin and eosin (HE) as well as
alcian blue and elastica-van-Gieson (AB-EVG) to detect cytoplasmic mucin production
and the elastic fiber framework. Lymphatic invasion and pulmonary metastasis were
evaluated on HE sections. Vascular and pleural invasion was evaluated in AB-EVG sections.
Sections were reviewed by 2 observers (M.O. and T.Y.) who were unaware of the clinical
data. Tumor size was measured as the maximal diameter on the cut sections of the lung.
Pathological stage was determined based on the criteria of the 7th TNM classification of Union of International Cancer Control [11].

Histological definition of micropapillary components

Histopathological diagnosis of lung adenocarcinoma was determined according to the
IASLC/ATS/ERS international multidisciplinary classification of lung adenocarcinoma
[1]. Comprehensive histological subtyping was performed on the primary tumor and divided
by percentage into 5 distinctive subtypes: lepidic, acinar, papillary, micropapillary,
and solid, totaling 100% per tumor. We defined the subtype as positive when it occupied
at least 1% of the entire tumor. We classified a micropapillary subtype into 2 components,
AMPC and SMPC, using the following criteria: AMPC is widely recognized in the lungs
as tumor cells floating within alveolar spaces, and SMPC includes papillary components
consisting of tufts lacking central fibrovascular cores, surrounded by lacunar spaces
and identified as invasive components in the stroma as previously described [9] (Figure 1A and 1B). Additionally, a tumor area without micropapillary components was defined as a non-micropapillary
component (nMPC).

Tumor tissue microarray (TMA) synthesis

TMAs were constructed using a manual tissue-arraying instrument (KIN-4; Azumaya, Tokyo,
Japan) as previously described [12], and specimens were punched using a stylet 3 mm in diameter.

Immunohistochemistry

The 17 antibodies used for immunohistochemical characterization of tumor cells in
TMA in this study are listed in Table 1. Immunohistochemical staining was performed as follows. TMA recipient blocks were
cut into 4-μm-thick sections and mounted on silane-coated slides. HE staining was
performed on initial sections to verify histology. The remaining sections were deparaffinized
in xylene and dehydrated in a graded alcohol series, and endogenous peroxidase was
blocked using 3% hydrogen peroxide in absolute methyl alcohol. Heat-induced epitope
retrieval was performed for 20 min at 95°C in 0.02 mol/L citrate buffer (pH 6.0) in
samples fixed with 10% formalin if necessary. The slides were rinsed using deionized
water and incubated with primary antibodies. They were then washed 3 times in phosphate-buffered
saline and incubated with EnVision+ System-HRP (DAKO, Glostrup, Denmark). The reaction
products were visualized using 3-3'-diaminobenzidine tetrahydrochloride, and sections
were counterstained using hematoxylin. Additionally, a similar staining method was
used for anti-podoplanin antibody (clone D2-40, pre-diluted; Ventana, Tucson, AZ,
USA) to evaluate lymphatic permeation.

Calculation of staining scores

Immunostaining was scored based on staining intensity and percentage of positively
stained cells, with 2 observers evaluating immunostained samples independently. When
the observers gave different scores to immunostained samples, the slides were reviewed
together under a multiheaded microscope until a consensus was reached. Sections were
classified by staining intensity as negative (total absence of staining), 1+ (weak
staining), 2+ (moderate staining), or 3+ (strong staining). Staining scores were calculated
by multiplying the percentage of positive tumor cells per section (0-100%) by the
staining intensity; scores obtained ranged from 0 to 300. Expression of p53, cleaved
caspase-3, and Ki-67 were determined by counting 300 tumor cells under a high power
field (×400) and results are shown as the percentage of positive cells.

Mutation analysis

Mutation analyses of EGFR gene exons 19 and 21 and KRAS gene codons 12 and 13 were performed using loop-hybrid mobility shift assays and gene
sequencing procedures described elsewhere [13].

Statistical analysis

All calculations were performed using SPSS software (Dr. SPSS II for Windows Standard
version 11.0; SPSS Inc., Chicago, IL, USA). The Chi-square for independence or Fisher's
exact probability test was performed to analyze differences in patient characteristics
between the 2 groups. The Fisher's exact probability test was performed if there were
5 or fewer observations in a group. For univariate analysis, all cumulative survival
was estimated using the Kaplan-Meier method, and differences in variables were calculated
using the log-rank test. Multivariate regression analysis was conducted according
to the Cox proportional hazard model. The Mann-Whitney U test was used to compare staining scores. Differences were considered significant
when the P value was less than 0.05.

Results

Clinicopathological characteristics of patients with SMPC

Figure 2 shows a Venn diagram of the relationship between the micropapillary component sets
in the 559 patients examined in this study. SMPC was observed in 19 patients (3.4%)
and AMPC in 99 (17.7%) patients. A mixture of SMPC and AMPC was observed in 14 patients,
pure SMPC without AMPC in 5 patients, and pure AMPC without SMPC in 85 patients. A
micropapillary pattern was observed in 50-100% in 2 SMPC tumor and less than 50% in
17 SMPC tumors. No SMPC(+) tumors were completely replaced by SMPC. Clinicopathological
characteristics of patients with SMPC(+) and SMPC(-) tumors are summarized in Table
2. Patients with SMPC(+) tumors were significantly found to be at a more advanced stage,
larger than 30 mm in diameter, and have more frequent lymph node metastasis compared
to those with SMPC(-) tumors. Pleural, lymphatic, and vascular invasion were observed
more often in patients with SMPC(+) tumors than in those with SMPC(-) tumors. (68%
vs. 17%, P < 0.001; 74% vs. 15%, P < 0.001; 74% vs. 22%, P < 0.001, respectively). No significant differences in age, gender, or smoking status
were observed between patients with SMPC(+) and SMPC(-) tumors.

Figure 2.Venn diagram of patients included in the present study. Among the 559 cases of lung adenocarcinoma, 104 cases had MPC. Nineteen cases had
SMPC (SMPC(+) tumors, the area enclosed by continuous line), and 99 had AMPC (AMPC(+)
tumors, the area enclosed by dotted line). A mixture of SMPC and AMPC was observed
in 14 patients, SMPC without AMPC in 5 and AMPC without SMPC in 85. MPC, micropapillary
component; SMPC, stromal micropapillary component; AMPC, aerogenous micropapillary
component.

Survival analysis

Among all stage patients, median follow-up time was 654 days (range, 33-1512 days)
in SMPC(-) tumors, 240 days (range, 28-661 days) in SMPC(+) tumors, 664 days (range,
28-1512 days) in AMPC(-) tumors, and 467 days (range, 36-1412 days) in AMPC(+) tumors.
Among the stage I patients, median follow-up time was 767 days (range, 59-1343 days)
in SMPC(-) tumors, 192 days (range, 227-485 days) in SMPC(+) tumors, 767 days (range,
59-1343 days) in AMPC(-) tumors, and 836 days (range, 140-1233 days) in AMPC(+) tumors.
Recurrence occurred in 28 of 559 cases. SMPC(+) tumors recurred in 4 of 19 in all
stage and in 2 of 10 in p-stage I, and AMPC(+) tumors recurred in 8 of 99 cases and
4 of 69 cases, respectively. In all stage, disease-free survival (DFS) of patients
with SMPC(+) tumors was significantly poorer than that in patients with SMPC(-) tumors
(Figure 3A, P < 0.001); the same result was observed in patients with AMPC(+) and AMPC(-) tumors
(Figure 3B, P = 0.045,). In p-stage I patients, DFS of those with SMPC(+) tumors showed significantly
poorer outcome than that of patients with SMPC(-) tumors (Figure 3C, P < 0.001); the same result was observed between patients with AMPC(+) and AMPC(-)
tumors (Figure 3D, P = 0.023).

Figure 3.Cumulative disease-free survival rates of patients according to presence of SMPC and
AMPC. A, B are cumulative disease-free survival rates in all stage, and C, D are that
of in p-stage I. Cumulative disease-free survival rates stratified by presence of
SMPC are shown in A and C, and those stratified by presence of AMPC are shown in B
and D. In all stage and in p-stage I, SMPC(+) tumors and AMPC(+) tumors had significantly
poorer outcomes. Outcomes of SMPC(+) tumors were more significantly negative than
those of AMPC(+) tumors. SMPC, stromal micropapillary component; AMPC, aerogenous
micropapillary component.

Table 3. Impact of potential prognostic factors on DFS of patients of lung adenocarcinoma in
all stage by univariate and multivariate analysis

Table 4. Impact of potential prognostic factors on DFS of patients of lung adenocarcinoma in
p-stage I by univariate and multivariate analysis

Immunohistochemical findings

We evaluated immunohistochemical profiles of SMPC, AMPC, and nMPC. These lesions were
evaluated in TMAs for 33 cases, including 19 SMPC(+) tumors and 14 pure AMPC tumors.
The latter 14 tumors were selected from 85 pure AMPC tumors according to operation
date, patient age, gender, and smoking status to match clinical background factors
between SMPC and AMPC. nMPC was generally included in TMA cores of SMPC and AMPC.
The total number of TMA was 19 SMPC and 28 AMPC. Staining scores are summarized in
Table 5.

In cellular adhesion molecules, E-cadherin staining scores in patients with SMPC,
AMPC, and nMPC were 215.3, 143.9, and 187.1, respectively, and although the differences
were not significant between patients with SMPC or nMPC and between patients with
AMPC or nMPC (P = 0.312, 0.127, respectively), staining scores of SMPC were significantly higher than
those for patients with AMPC (P = 0.020) (Figure 4A-C). CD44 staining scores in SMPC, AMPC, and nMPC were 60.8, 205.9, and 141.3, respectively.
The CD44 expression level in SMPC was significantly lower than in AMPC (P < 0.001) and significantly higher than that in nMPC lesions (P = 0.015) (Figure 4D-F).

For other antibodies, staining scores of surfactant apoprotein A (SP-A) in the SMPC,
AMPC, and nMPC were 45.2, 82.6, and 123.2, respectively, and although the difference
was not significant between AMPC and nMPC (P = 0.203), the staining score in SMPC was significantly lower than those in nMPC (P = 0.024) (Figure 4G-I). Similarly, staining scores of phospho-c-Met in SMPC, AMPC, and nMPC were 34.2,
50.5, 88.0, respectively, and staining scores in SMPC were significantly lower than
those in nMPC (Figure 4J-L).

Mutation analysis

Mutation analysis was performed in 33 patients for whom TMAs were constructed for
immunohistochemical analysis. Table 6 summarizes the results of the mutation analysis. Although no cases examined possessed
the KRAS mutations, EGFR mutations were detected in 20 cases (61%): 14 in patients with SMPC(+) tumors (74%)
and 6 in patients with SMPC(-) tumors (43%). There was no significant association
between the existence of SMPC and EGFR mutations. Among the 20 cases with EGFR mutations, 7 had deletions at exon 19, 13 had a point mutation at exon 21, and there
were no multiple mutations. Among the 13 cases with a point mutation at exon 21, 12
had an L858R mutation and one had an L861Q mutation.

Discussion

The present study revealed the incidence of SMPC(+) lung adenocarcinoma in consecutive
surgical cases to be 3.4%, which is lower than that of AMPC(+) lung adenocarcinoma
(17.7%). In non-pulmonary organs, the incidence of invasive micropapillary carcinoma
was reported to be 7% in breast carcinoma [14], 0.9% in urinary bladder cancer [15], and 9.4% in colon cancer [16]. Generally, invasive micropapillary carcinomas occur infrequently in any organ.

Prognosis of lung adenocarcinoma with MPC has been reported to be worse and have the
potential for high malignancy [17,18], but no studies have separately evaluated SMPC and AMPC. We showed that SMPC(+) tumors
as well as AMPC(+) tumors are associated with several biological factors including
tumor size, lymph node metastasis, advanced stage disease, and pleural and lymphovascular
invasion. Univariate analysis also revealed the presence of SMPC and AMPC as a significant
predictor of unfavorable outcome. However, the most remarkable finding was observed
in multivariate analysis: among the patients in p-stage I, patients with not AMPC
but SMPC showed a significantly poorer DFS than those without MPC. We used immunohistochemistry
with monoclonal antibody D2-40 against lymphatic endothelium in TMA specimens and
found that lymphatic vessels are involved within SMPC areas in 4 (21%) of 19 SMPC(+)
tumors (data not shown). When compared with AMPC(+) tumors, SMPC(+) tumors significantly
more often showed pleural, lymphatic, and vascular invasion than AMPC(+) tumors (68%
vs. 33%, P = 0.004; 74% vs. 30%, P < 0.001; 74% vs. 41%, P = 0.010, respectively). Therefore, these data suggest that a strong association between
SMPC(+) tumors and pleural and lymphovascular invasion may in part explain their aggressive
behavior.

Moreover, we investigated the immunohistochemical differences between SMPC and AMPC.
In the study, we observed high E-cadherin expression and low CD44 expression in SMPC.
Phospho-c-Met expression generally decreases in SMPC to a greater extent than in AMPC.
Recently, it has been suggested that E-cadherin repression and CD44 expression are
associated with the epithelial-mesenchymal transition (EMT), which was thought to
lead to tumor invasion [19,20]. Additionally, Elliot et al. reported that hepatocyte growth factor (HGF) and c-Met
signaling promotes EMT in breast cancer [21], and Orian-Rousseau et al. reported that CD44 is strictly required for c-Met activation
by HGF in human carcinoma [22]. Consistent with these data, EMT may not occur in SMPC despite its existence in the
stroma, or invasion of SMPC may occur through a different invasion mechanism from
EMT. Our immunohistochemical findings of SMPC showed lower expression of SP-A than
that of nMPC. Many studies have reported that SP-A deletion is correlated with patient
survival, and reduced SP-A in MPC may be an excellent indicator for poor prognosis
in small-size lung adenocarcinoma [23,24]. Reduced SP-A may contribute to an unfavorable outcome of SMPC(+) tumors.

Some studies have reported a significant association between the presence of MPC and
EGFR mutations and effectiveness of EGFR tyrosine kinase inhibitor (EGFR-TKI) for MPC(+)
tumors [25-28]. Since SMPC of lung adenocarcinoma may be associated with a high incidence of EGFR mutations, EGFR-TKI may be effective against SMPC(+) tumors. Patients with these pathological
features of lung adenocarcinoma may benefit from EGFR-TKI as postoperative chemotherapy
or first-line chemotherapy of relapsed lung adenocarcinoma.

In conclusion, we observed SMPC(+) adenocarcinoma. The incidence of SMPC(+) tumors
is low, and SMPC(+) tumors have a different prognostic impact compared to AMPC(+)
tumors. Particularly for the early stage tumors, SMPC(+) tumors have different pathobiological
characteristics from AMPC(+) tumors, and SMPC(+) tumors frequently contain the EGFR mutation. Therefore, it is important to determine the presence of SMPC in lung adenocarcinoma,
particularly p-stage I tumors, and the presence of SMPC should be noted in a pathology
report to alert the clinician to the possibility of poor prognosis.

List of abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MO and TY designed the study, performed clinical and pathological investigation, and
wrote the drafts. YS and YM participated in pathological and genetical investigation.
NO participated in statistical investigation. SO performed the histological and immunohistochemical
evaluation. CH assisted the clinical investigation. HN participated in managing and
operating the patients. YK assisted the pathological investigation. KY participated
in collecting clinical data and images. TI participated in its design and coordination
and helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Ms. Yoshihara for TMA synthesis.

This article was supported by Kanagawa Cancer Research Fund and Grant for Collaboration
between Hospital and Research Institute, Kanagawa Cancer Center.